Supplemental Figure 2. Histological evaluation of tumor progression and confirmation of IDO-mGFP expression in brain tumors.

Supplementary Table 4. Mass spectrometry parameters for detecting CFH and FHL-1 in plasma.

Supplemental Figure 1. Overall survival of syngeneic mice with intracranial IDO-/-tGBM tumors and depleted for CD4+ T, CD8+ T, and NK1.1+ immune cells.

Supplementary Table 2. Nucleic acid-based reagents utilized.

Supplementary Table 1. Demographics of patient-isolated tissue samples.

Supplementary Table 3. Differentially expressed genes that possess the strongest correlation with IDO in human GBM cells.

TS and hENT1 expression are not altered 6 h after gemcitabine.

Supplementary Methods from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

one of the most highly recognized scientists in this field. His pioneering studies included deciphering the metabolic interactions between neurons and glial cells in the adult brain, the development of multinuclear spectroscopy methods for metabolic studies, and more recently the development of novel imaging and spectroscopy approaches to investigate the brain in obesity. Sebastián's studies with 13 C labeled substrates such as 13 C pyruvate, 13 C glucose, and 13 C glutamate in experimental studies of animals drinking deuterated water served as precursors for the 13 C and deuterium labeling studies of today. Sebastián developed, patented, and transferred to industry an extensive collection of novel probes to noninvasively monitor extracellular pH, novel Gd(III) complexes, nanostructured tubular graphene derivatives, and magneto-liposomes with improved performance as contrast agents for diagnostic MRI.

Over the last half century, there have been several periods during which magnetic resonance spectroscopy (MRS) has been used ex vivo, for a variety of reasons, on samples such as microorganisms, cells, animal or human tissue, tissue extracts or biological fluids. These studies began in the days before the acronym MRS had been invented, when all such methods were still called nuclear magnetic resonance (NMR), and have extended to the present day. I will describe the historical development of NMR methods used ex vivo, their influences on the development of MRS in vivo, and their longer-term uses. All the interpretations will be personal, based on what I saw, or discussed with colleagues at the time.

Purpose: Glioblastoma (GBM) is an incurable primary brain tumor that has not benefited from immunotherapy to date. More than 90% of GBM expresses the tryptophan (Trp) metabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO). This observation supported the historical hypothesis that IDO suppresses the antitumor immune response solely through a mechanism that requires intratumoral Trp depletion. However, recent findings led us to investigate the alternative hypothesis that IDO suppresses the anti-GBM immune response independent of its association with Trp metabolism.Experimental Design: IDO-deficient GBM cell lines reconstituted with IDO wild-type or IDO enzyme-null cDNA were created and validated in vitro and in vivo. Microarray analysis was conducted to search for genes that IDO regulates, followed by the analysis of human GBM cell lines, patient GBM and plasma, and The Cancer Genome Atlas (TCGA) database. Ex vivo cell coculture assays, syngeneic and humanized mouse GBM models, were used to test the alternative hypothesis.Results: Nonenzymic tumor cell IDO activity decreased the survival of experimental animals and increased the expression of complement factor H (CFH) and its isoform, factor H like protein 1 (FHL-1) in human GBM. Tumor cell IDO increased CFH and FHL-1 expression independent of Trp metabolism. Increased intratumoral CFH and FHL-1 levels were associated with poorer survival among patients with glioma. Similar to IDO effects, GBM cell FHL-1 expression increased intratumoral regulatory T cells (Treg) and myeloid-derived suppressor cells while it decreased overall survival in mice with GBM.Conclusions: Our study reveals a nonmetabolic IDO-mediated enhancement of CFH expression and provides a new therapeutic target for patients with GBM.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 μg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 μg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 μg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

Topographical variations of metabolite concentrations have been reported in the duodenum, jejunum and ileum of the small intestine, and in human intestinal tumours from those regions, but there are no published metabolite concentrations measurements correlated with linear position in the mouse small intestine or intestinal tumours. Since DNA methylation dynamics are influenced by metabolite concentrations, they too could show linear anatomical variation. We measured metabolites by HR-MAS 1 H NMR spectroscopy and DNA cytosine modifications by LC/MS, in normal small intestines of C57BL/6J wild-type mice, and in normal and tumour samples from Apc Min/ + mice. Wild-type mouse intestines showed approximately linear, negative concentration gradations from the pylorus (i.e. the junction with the stomach) of alanine, choline compounds, creatine, leucine and valine. Apc Min/ + mouse tumours showed negative choline and valine gradients, but a positive glycine gradient. 5-Hydroxymethylcytosine showed a positive gradient in the tumours. The linear gradients we found along the length of the mouse small intestine and in tumours contrast with previous reports of discrete concentration changes in the duodenum, jejunum and ileum. To our knowledge, this is also the first report of a systematic measurement of global levels of DNA cytosine modification in wild-type and Apc Min/+ mouse small intestine.

In recent years the use of NMR methods to detect and quantify metabolites in body fluids (urine, blood, cerebrospinal fluid etc) has become widespread. There has been a lot of exciting research in this area, so we have decided that that NMR in Biomedicine will publish papers on these topics, and we will accept such manuscripts for review in the usual way. Prof Tone Frost Bathen, who has recently joined our Editorial Advisory Board, will cover this field in conjunction with Prof Leo Cheng. The journal's Aims and Scope will now read as follows:

Mass spectrometry (MS) is a sensitive analytical technique with wide application across the sciences including for the detection of peptides and proteins in biological analysis. Ubiquitinated (Ub) proteins are typically analyzed by proteolytic digestion and subsequent chromatographic separation followed by MS detection of the resulting isopeptides. Here we describe a novel method which enables enhanced detection of this important posttranslational modification (PTM) by use of a simple chemical labeling strategy prior to Data-Independent Acquisition (DIA) using a SWATH-based acquisition approach on a suitable Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer.

1 European Institute for Molecular Imaging (EIMI), Westfälische Wilhelms-Universität Münster, Münster, Germany 2 Department of Clinical Radiology, University Hospital of Münster, Münster, Germany 3 Roche Pharma Research and Early Development, Pathology and Tissue Analytics, Roche Innovation Center Munich, Germany 4 Comprehensive Cancer Imaging Centre, Imperial College London, UK 5 Cancer Research UK Cambridge Institute, Cambridge, UK 6 Laboratory of imaging biomarkers, UMR 1149 CRI, Inserm, Paris Diderot University, Paris, France 7 Imaging Sciences & Biomedical Engineering Division Kings College, London, UK 8 Merck KGaA, Darmstadt, Germany 9 Department of Geriatric Medicine, Johanniter Hospital, Bonn, Germany *equal contribution

NMR in BiomedicineVolume 32, Issue 1 e4047 EDITORIAL Membership of the Editorial Advisory Board John Griffiths, Corresponding Author John Griffiths Editor-in-Chief John.Griffiths@cruk.cam.ac.uk Search for more papers by this author John Griffiths, Corresponding Author John Griffiths Editor-in-Chief John.Griffiths@cruk.cam.ac.uk Search for more papers by this author First published: 04 December 2018 https://doi.org/10.1002/nbm.4047Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume32, Issue1January 2019e4047 RelatedInformation

NMR in BiomedicineVolume 31, Issue 7 e3953 EDITORIAL NMR in Biomedicine 30th Anniversary Volume Message from the Editor-in-Chief John Griffiths, Corresponding Author John Griffiths john.griffiths@cruk.cam.ac.uk Cancer Reasearch UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, United Kingdom Correspondence John Griffiths, Cancer Reasearch UK Cambridge Institute, Li Ka Shing Centre, Robinson Way,Cambridge CB2 0RE, United Kingdom, United Kingdom. Email: john.griffiths@cruk.cam.ac.ukSearch for more papers by this author John Griffiths, Corresponding Author John Griffiths john.griffiths@cruk.cam.ac.uk Cancer Reasearch UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, United Kingdom Correspondence John Griffiths, Cancer Reasearch UK Cambridge Institute, Li Ka Shing Centre, Robinson Way,Cambridge CB2 0RE, United Kingdom, United Kingdom. Email: john.griffiths@cruk.cam.ac.ukSearch for more papers by this author First published: 24 May 2018 https://doi.org/10.1002/nbm.3953Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume31, Issue7July 2018e3953 This article also appears in:NMR in Biomedicine: 30 Year Celebration RelatedInformation

Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.